Preparation of templates for nucleic acid sequencing
The invention relates to methods of generating templates for a nucleic acid sequencing reaction which comprise: providing at least one double-stranded nucleic acid molecule, wherein both strands of the double-stranded nucleic acid molecule are attached to a solid support at the 5′ end, cleaving one or both strands of the double-stranded nucleic acid molecule, and subjecting the cleaved strand(s) to denaturing conditions to remove the portion of the cleaved strand(s) not attached to the solid support, thereby generating a partially or substantially single-stranded template for a nucleic acid sequencing reaction.
1. A method of preparing single-stranded templates for a nucleic acid sequencing reaction comprising,
(i) providing a solid support comprising a plurality amplification primers, wherein a subset of said plurality of amplification primers comprises a cleavage site;
(ii) amplifying a template using the subset of primers on the support to produce a plurality of double-stranded nucleic acid molecules, wherein both strands of each double-stranded nucleic acid molecule are attached to the solid support at their 5′ ends, whereby the cleavage site is positioned in a double-stranded region of each double-stranded molecule;
(iii) cleaving only one strand of the double stranded molecules at the cleavage site, the cleavage not comprising cleavage with a restriction endonuclease or a nicking endonuclease;
(iv) subjecting the cleaved strand to denaturing conditions to remove the portion of the cleaved strand not attached to the solid support, followed by re-annealing the cleaved strand to the opposite strand, thereby generating immobilized partially or substantially single-stranded templates; and
(v) hybridizing a sequencing primer to the immobilized partially or substantially single-stranded templates, thereby preparing single-stranded templates for a nucleic acid sequencing reaction.
2. The method of claim 1 , further comprising performing a sequencing reaction to determine the sequence of at least one region of the immobilized single-stranded templates.
3. The method of claim 1 , further comprising treating the cleaved strand with a capping agent prior to step (v).
4. The method of claim 3 , wherein said cleaving and treating with a capping agent occur concurrently.
5. The method of claim 3 , wherein said cleaving and treating with a capping agent occur non-concurrently.
6. The method of claim 1 , wherein cleavage comprises cleaving via a chemical cleavage reaction.
7. The method of claim 1 , wherein cleavage comprises generating an abasic site at the cleavage site.
8. The method of claim 1 , wherein the cleavage site comprises a uracil.
9. The method of claim 1 , wherein the cleavage site comprises 8-oxo-guanine.
10. The method of claim 1 , wherein the cleavage site comprises deoxyinosine.
11. The method of claim 1 , wherein the cleavage site comprises one or more ribonucleotides.
12. The method of claim 1 , wherein cleavage occurs by exposure to a metal ion.
13. The method of claim 1 , wherein the cleavage site comprises one or more methylated nucleotides.
14. The method of claim 1 , wherein cleavage occurs by a photochemical mechanism.
15. The method of claim 1 , wherein amplification of the template forms a cluster comprising the plurality of double-stranded molecules.
16. The method of claim 1 , wherein the sequencing reaction comprises sequencing-by-synthesis.
17. The method of claim 1 , wherein the sequencing reaction comprises pyrosequencing or sequencing-by-ligation.
18. The method of claim 1 , wherein the solid support comprises a hydrogel.
19. The method of claim 18 , wherein the hydrogel is a polyacrylamide hydrogel.
20. The method of claim 18 , wherein the solid support is prepared by a method comprising polymerizing on the solid support a mixture of:
(i) a first comonomer which is acrylamide, methacrylamide, hydroxyethyl methacrylate or N-vinyl pyrrolidinone; and
(ii) a second comonomer which is a functionalized comonomer selected from acrylamide or acrylate of formula (I):
H2C═C(H)—C(═O)-A-B-C (I);
or a methacrylate or methacrylamide of formula (II):
H2C═C(CH3)—C(═O)-A-B-C (II);
wherein
A is NR or O, wherein R is hydrogen or an optionally substituted saturated hydrocarbyl group comprising 1 to 5 carbon atoms;
B is an optionally substituted alkylene biradical of formula —(CHn)- wherein n is an integer from 1 to 50; and wherein n=2 or more, one or more optionally substituted ethylene biradicals —CH2CH2- of said alkylene biradical may be independently replaced by ethenylene and ethynylene moieties; and wherein n=1 or more, one or more methylene biradicals —CH2- may be replaced independently with an optionally substituted mono- or polycyclic hydrocarbon biradical comprising from 4 to 50 carbon atoms, or a corresponding heteromonocyclic or heteropolycyclic biradical wherein at least 1 CH2 or CH2 is substituted by an oxygen sulfur or nitrogen atom or an NH group; and
C of A-B-C of formulas (I) or (II) is a group for reaction with a compound to bind said compound covalently to said hydrogel to form a polymerized product,
characterized in that polymerization is conducted on and immobilized the polymerized product to a solid support that is not covalently surface-modified.
21. The method of claim 1 , wherein the plurality of amplification primers comprises a set of forward amplification primers and a set of reverse amplification primers, and wherein the subset comprising the cleavage site is the set of forward primers.
22. The method of claim 1 , wherein the plurality of amplification primers comprises a set of forward amplification primers and a set of reverse amplification primers, and wherein the subset comprising the cleavage site is the set of reverse primers.